Many regulatory proteins are heterodimers, that is, they are composed of two different peptide chains which interact to generate the native protein.
Among such regulatory proteins are DNA binding proteins which are capable of binding to specific DNA sequences and thereby regulating transcription of DNA into RNA. The dimerization of such proteins is necessary in order for these proteins to exhibit such binding specificity. A large number of transcriptional regulatory proteins have been identified: Myc, Fos, Jun, Ebp, Fra-1, Jun-B, Sp1, H2TF-1/NF-.kappa.B-like protein, PRDI, TDF, GLI, Evi-1, the glucocorticoid receptor, the estrogen receptor, the progesterone receptor, the thyroid hormone receptor (c-erbA) and ZIF/268, OTF-1(OCT1), OTF-2(OCT2) and PIT-1; the yeast proteins GCN4, GAL4, HAP1, ADR1, SWI5, ARGRII and LAC9, mating type factors MAT.alpha.1, MATa2 and MATa1; the Neurospora proteins cys-3 and possibly cpc-1; and the Drosophila protein bsg 25D, kruppel, snail, hunchback, serendipity, and suppressor of hairy wing, antennapedia, ultrabithorax, paired, fushi tarazu, cut, and engrailed. Eukaryotic transcriptional regulatory proteins, and the methods used to characterize such proteins, have been recently reviewed (Pabo, C.O. et al., Ann. Rev. Blochem. 61:1053-1095 (1992); Johnson, P. F. et al., Ann. Rev. Blochem. 58:799-839 (1989)).
Members of the mammalian transcriptional regulatory protein families Jun/Fos and ATF/CREB only bind to DNA as dimers. The proteins in these families are "leucine zipper" proteins which contain a region rich in basic amino acids followed by a stretch of about 35 amino acids which contains 4-5 leucine residues separated from each other by 6 amino acids (the "leucine zipper" region). Collectively, the combination of a basic region and the leucine zipper region is termed the bZIP domain.
Generally, it is the basic region which has been found to be predominantly involved in contacting DNA whereas the zipper region mediates the dimerization. Many dimeric combinations are possible, however, the particular nature of the zipper specifies which partnerships are permissible (Abel, T. et al., Nature 341:24-25 (1989)).
Another large family of proteins contains the DNA binding/dimerization motif known as the basic helix-loop-helix motif (bHLH) (Jones, N., Cell 61:9-11 (1990)). A bHLH protein generally contains a basic N-terminus followed by a helix-loop-helix structure; two short amphipathic helices containing hydrophobic residues at every third or fourth position. The sequence of the basic region characteristically reveals no indication of an amphipathic helix. The intervening loop region usually contains one or more helix-breaking residues.
The bHLH motif was first detected in two proteins, E12 and E47, that bind to a specific "E box" DNA enhancer sequence found in immunoglobulin enhancers (Murre C. et al., Cell 56:777-783 (1989)). E motifs generally are double stranded variants of the 5'-CAGGTGGC-3'consensus sequence. For example, the .mu.E1 motif is GTCAAGATGGC [Seq. ID NO. 1], .mu.E2 motif is AGCAGCTGGC [SEQ ID NO. 2], .mu.E3 is GTCATGTGGC [Seq. ID NO. 3], .mu.E is TGCAGGTGT (Murre, C. et al., Cell 56:777-783 (1989)). Like many transcriptional factors, peptides containing the bHLH motif often dimerize with each other, either as a homodimer which contains two identical peptides or as a heterodimer which contains two different peptides. Examples of heterodimeric complex of two bHLH proteins binding DNA with a greater efficiency than homodimeric complexes of either peptide in the heterodimer are known (Murre C. et al., Cell 56:777-783 (1989); Murre, C et al., Cell 58:537-544 (1989)).
Identification of partners which direct protein-DNA binding and compounds which inhibit such activity by inhibiting such protein partner interaction could be very useful. For example, identification of partners of the myc protein and inhibitors of myc-partner interactions could provide a means for treating diseases in which expression and activity of myc is a factor in promoting cell growth or in maintaining the cell in a transformed state.
Myc is a bHLH protein and the bHLH domain of c-myc is encoded in c-myc amino acids 354-411. The sequence homology between the proteins expressed by the three myc genes (human N-myc 393-437, human c-myc 354-411, and human L-myc 289-338) and other genes which contain a bHLH domain have been compared (Murre C. et al., Cell 56:777-783 (1989)).
Proteins such as myc which contain the bHLH motif also possess the ability to dimerize with other bHLH motif proteins. Such interactions among bHLH proteins may play a critical role in their function and/or regulation. Identification of these protein partners would be useful not only in understanding how these proteins function, but also in developing or identifying inhibitors of these proteins. For example, identification of myc-partners would make it possible to identify inhibitors of myc-partner interactions. By inhibiting such interactions, inhibition and/or control of myc-induced cell growth may be achieved.
To date, no myc inhibitors have been identified. The identification of such inhibitors has suffered for lack of a simple, inexpensive and reliable screening assay which could rapidly identify potential inhibitors and active derivatives thereof. Thus a need still exists for rapid, economical screening assays which identify specific inhibitors of oncogene activity.